Studies of innate immune responses have been highly influenced by the discovery of the IKKrelated kinases, TBK1 and IKKe. TBK1 and IKKe substrates include the transcription factors interferon regulatory factor (IRF)-3 as well as the closely related IRF7. This proposal will investigate the molecular mechanisms underlying TBK1-dependent production of antiviral cytokines including type 1 interferons (IFN). We provide preliminary data that identify two E3 ubiquitin ligases which target TBK1 for K63-linked polyubiquitination (pUb), regulate NF-kB and IRF3 signaling, and control viral infection. In Specific Aim 1 we analyze TBK1 pUb. This includes characterization of the E3 ligases and identification of the TBK1 Ub acceptor site. As background we provide evidence that TBK1 is posttranslationally modified by K63-linked pUb chains. We also supply preliminary data indicating a role for mindbomb (MIB) proteins in controlling the levels of TBK1 pUb and IFN production. This is the first evidence that the E3 ligases MIB1 and MIB2 participate in IRF dependent responses. We will determine the functional significance of TBK1 pUb in activation of IRF3 and in viral infection. Specific Aim 2 will investigate the consequences of MIB1 and/or MIB2 deficiency on IFN production and protection against viral replication. Initial experiments have reassuringly supported a role for mib genes in protection against vesicular stomatitis virus (VSV). Specific Aim 3 will evaluate specificity requirements for TBK1 pUb. We will determine the dependency of MIB-mediated pUb in activation of other IKK kinases, examine TLR4 responses to bacterial lipopolysaccharide (LPS), and investigate clearance of various RNA and DNA viruses by fibroblasts, macrophages, and dendritic cells. The final Specific Aim identifies TBK1 interacting proteins and investigates how TBK1 pUb participates in assembly of the signalosome. Detailed descriptions of the molecular mechanisms that regulate TBK1 activity are important since dysregulation of this kinase is associated with defects in host defense to viral infection. The insights from such studies are ultimately needed to design therapies that may curtail IKKrelated pathways in chronic inflammatory diseases or enhance TBK1 activity when needed to boost immunity, especially for virally induced pathologies.